In nonmuscle cells, myosin mediates several functions including cytokinesis, granular secretion and shape change. For example, studies using rat basophil leukemic cells (RBL-2H3) have shown that phosphorylation of myosin is associated with secretion of histamine. It is also known that treatment of lymphocytes with Interleukin 2 (IL-2) leads to cell proliferation as well as augmentation of a variety of lymphocyte activities such as migration and secretion. The goals of this study are to examine in vivo phosphorylation of myosin in human lymphocytes, to identify the kinase(s) involved in phosphorylation of myosin and to understand the physiological significance of this phosphorylation. We have shown that treatment of 32P-labeled Jurkat cells with phorbol ester (PMA) results in incorporation of phosphate in both the myosin heavy chain and the 20 kD light chains. Isoelectric focusing of phosphorylated myosin heavy chain showed a new phosphopeptide in PMA treated cells suggesting the activation of protein kinase C. We also examined phosphorylation of myosin in CD16-enriched lymphocytes, isolated from buffy coats obtained from healthy donors. Short-term treatment of these cells with IL-2, a physiological activator, led to incorporation of phosphate into myosin heavy and light chains. Isoelectric focusing of the tryptic phosphopeptides reveals that the increase in phosphorylation of myosin light chains is due to the increased activity of myosin light kinase and protein kinase C. In the case of myosin heavy chain, the increase in phosphorylation may be due to the casein kinase II. We have shown that when CD16 cells are treated with PMA, the isoelectric focusing of the tryptic phosphopeptides was identical to those from the Jurkat cells treated with PMA. These results suggest that the short-term stimulation with IL-2 was not associated with protein kinase C activation in CD16 cells. We will continue this study by correlating myosin phosphorylation with physiological function such as ATPase activity and in vitro motility assays as well as in situ functions such as lymphocyte degranulation. Finally, we wish to compare the phosphorylation of the myosin heavy chain from lymphocytes which contain mostly the myosin heavy chain-A isoform with the myosin heavy chain from bovine brain which is primarily the B isoform.